Hole in the Wall Offers Cheaper Sequencing

Today’s gene-sequencing labs are scientifically luxurious establishments, replete with expensive reagents and high-tech equipment. But if Daniel Branton and David Deamer have their way, these ritzy facilities might one day be replaced by something much more like a hole in the wall.

Not just any hole in the wall: The one they have in mind is a pore just wide enough to permit a single strand of DNA or RNA to pass through. As the genetic material runs through the tiny portal like a very long train through a very short tunnel, the scientists hope to discern the sequence of its subunits in real time.

The pore-based approach marks a drastic departure from conventional techniques, which rely on a complicated series of steps to prepare large quantities of DNA and then deduce the sequence of subunits (bases) indirectly. By directly reading bases, the pore system could cut the cost and time of sequencing.

“It’s very appealing new technology,” says Charles Cantor, a Boston University biophysicist and former principal scientist for the Department of Energy’s Human Genome Project. “In a lot of DNA sequencing projects, the sample preparation is becoming the cost-limiting step,” says Cantor. “If you could do single-molecule sequencing, then it’s quite possible you could dramatically cut down the burden of sample preparation.”

In 1991 Deamer and Branton realized that because DNA and RNA are electrically charged, it ought to be possible to use an electric field to drive them through a pore embedded in a thin membrane. As individual strands traverse the membrane, they would partially block the channel and cause a drop in the current. Each of the four bases in DNA or RNA is a different size, so each should block the channel in a slightly different way; by monitoring the current, the scientists could-at least in theory-read the molecule’s sequence.

Now Deamer, a biophysicist at the University of California, Santa Cruz, and Branton, a cell biologist at Harvard, are heading a cross-country collaboration to turn the strategy into a working system. Their first target is RNA, whose subunits are conventionally referred to by the letters A,U, G, and C. Using a channel-shaped protein normally made by bacteria as the pore, Deamer and Branton have, so far, been able to recognize the electrical signal from RNA molecules with a simple sequence of 70 As followed by 30 Cs. The researchers estimate that eventually they could read about 1,000 bases a second, more than 500 times faster than the leading automatic sequencing device. Though the resolution isn’t yet fine enough to distinguish individual bases, Branton says, “what we have here is proof of principle.”

Jeffery Schloss, director of the National Human Genome Research Institute’s sequencing technology program, says that though the system is “pretty far from realization,” it is “very intriguing.”

Others are also working on single-molecule sequencers. One approach uses atomic-force microscopes to “see” sequences directly; another employs fluorescence detectors to read labeled bases clipped one at a time from the end of a DNA strand. But the pore system requires less sample preparation and expensive equipment than such methods.

Deamer and Branton will continue trying to optimize the pore-based approach on opposite coasts. Tuning the system to read single bases will be a daunting task, but the researchers take heart from their recent success. Says Deamer: “It gives us a lot of confidence that we’re on the right track.” And that track might one day lead them to a very classy little hole in the wall.